Effects of 10(fold upregulation).

*<p>p<0.05.</p>**<p>p≤0.01.</p

Effects of 1,25(OH)₂D on RV replication.

<p>(A) Replication of RV16 at 24 hours in differentiated hBEC cultured at air-liquid interface for 24 days in the presence of various concentrations of 1,25(OH)₂D (n = 7). (B) Replication of RV16 (MOI = 0.1, 1, or 10) at 24 hours in differentiated hBEC cultured at air-liquid interface for 27 days in the presence of 10 nM of 1,25(OH)₂D (n = 3).</p

Effects of 1,25(OH)₂D on cell morphology.

<p>(A) Hematoxylin and eosin stained paraffin cross-sections of primary hBEC layers cultured at an air-liquid interface for 24 days in the presence of 10 nM 1,25(OH)₂D. (B) Staining of paraffin cross-sections from 1,25(OH)₂D-treated primary hBEC cell layers with antibody against cytokeratin 5/6 (CK5/6). (C) Alcian blue stained paraffin cross-sections of primary hBEC layers cultured at an air-liquid interface for 24 days in the presence of 10 nM 1,25(OH)₂D. (D) Average thicknesses of primary hBEC layers differentiated in the presence of 10 nM 1,25(OH)₂D for 4 to 6 weeks (n = 8). (E) Average thicknesses of fully differentiated primary hBEC layers after treatment with 10 nM 1,25(OH)₂D for 6 weeks (n = 5).</p

Effects of 1,25(OH)₂D on cytokine secretion.

<p>(A,B) 1,25(OH)₂D enhanced RV-induced CXCL8 (n = 6), and CXCL10 (n = 5) secretion. (C–E) Production of CCL5, IL-29, and IL-6 (n = 6) in these same cells, with “n.d.” indicating non-detectable levels. *p≤0.05 for cells treated with 1,25(OH)₂D compared to untreated cells.</p

1,25(OH)₂D and 25(OH)D have similar effects on cell growth and differentiation.

<p>Hematoxylin and eosin stained paraffin cross-sections of primary hBEC layers cultured at an air-liquid interface for 28 days in the presence of various concentrations of 1,25(OH)₂D or 25(OH)D.</p

Similar effects of supplemental vitamin D vs. retinoic acid deficiency on cell morphology.

<p>Hematoxylin and eosin-stained paraffin cross-sections of primary hBEC layers cultured at an air-liquid interface for 28 days in the presence of 10 nM (deficient) or 50 nM (normal) all-<i>trans</i> retinoic acid, with 0 nM or 10 nM 1,25(OH)₂D.</p

Nucleotide sequence changes required to switch H5 HA receptor binding preference.

<p>(A) Analysis of codon usage by 1374 H5 influenza viruses indicates that codon 228 is GGA in 93% strains and GGG in 7%. The table illustrates transversions and transitions by which coding capacity at this residue might change from glycine to serine. The ability of the two intermediate mutants 228R (B) and 228A (E) recombinant HA proteins to bind synthetic receptor ligands 3SLN (avian receptor) and 6SLN (human receptor) in a solid phase assay was assessed. The binding phenotype on HAE (C,F) or human tracheal epithelium (D,G) by recombinant proteins with each amino acid sequence changed at residue 228 are shown (C,D) G228R and (F,G) G228A.</p

Expression of recombinant HA-Fc proteins.

<p>(A) Recombinant baculoviruses encoding HA from a recent H3N2 human virus, (A/Panama/2007/99) or from a highly pathogenic H5N1 avian influenza virus (A/Vietnam/1194/04), were generated as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007836#pone.0007836-Barclay1" target="_blank">[32]</a>. The HA proteins were expressed as soluble proteins secreted from infected <i>Sf9</i> cells by removing the HA transmembrane (TM) and cytoplasmic tail (CT) portions of the protein and replacing the HA signal peptide (SP) with the signal peptide of the baculovirus envelope protein gp64 (SP gp64). The HA proteins were tagged at the C-terminus by a human Fc (HuFc) and hexa-histidine (His6) tags. (B) All recombinant HA proteins were expressed at similar levels as determined by western blot analysis using an anti-human Fc antibody.</p

Recombinant HA proteins from human and avian influenza viruses bind <i>ex vivo</i> human tracheal epithelium in a sialic acid dependant manner.

<p>Histological sections of <i>ex vivo</i> human tracheal epithelium were probed with human H3 (A), avian H5 (B) or H5 mutants, (C) G228S, and (D) Q226L/G228S, with and without prior neuraminidase (NA) treatment. Slides were pre-incubated for 3 hours with 5 units of recombinant NA cloned from <i>Salmonella typhimurium</i> LT2 (NEBL) that shows a 260-fold preference for α2-3 over α2-6 linked SA or with 15 units of recombinant NA cloned from <i>Clostridium perfringens</i> (NEBL) that cleaves both α2-3 and α2-6 SA linkages. HA was detected as before using anti-human Fc (red) and anti-acetylated α-tubulin was used to indicate ciliated cells (green). White arrows indicate cells that exhibit HA binding. Open arrowheads indicate ciliated cells with HA binding; solid arrowheads indicate non-ciliated cells with HA binding.</p

Binding of recombinant HA proteins to synthetic receptor ligands 3SLN (avian receptor) and 6SLN (human receptor) in a solid phase assay.

<p>(A) Synthetic glycans on polyacrylamide linkers were immobilized on 96-well plates following UV treatment. Recombinant HA proteins were preformed into higher order complexes by incubation with anti-human Fc before incubation on the plate and detection with goat anti-human IgG conjugated to horse-radish peroxidise (HRP). (B) Recombinant HA-Fc proteins were adsorbed on 96 well plates coated with anti-human Fc antibody. Synthetic glycans on polyacrylamide linkers with biotin tags (6SLN and 3SL) were incubated on the plates and detected with Streptavidin – HRP conjugate.</p